DNA mismatch repair (MMR) is a major contributor to genome stability. MMR corrects DNA biosynthetic errors, ensures the fidelity of genetic recombination and is required for the cellular response to certain classes of DNA damage, including lesions induced by several chemotherapeutic drugs (e.g. cisplatin). MMR also has an essential role in somatic hypermutation for the generation of immunoglobin diversity. Defects in human MMR are associated with a strong predisposition to tumor development, and mutagenic expansion of CTG-CAG repeat sequence which are the causative mutations for several meuromuscular diseases (Huntington's disease, fragile-X syndrome, and myotonic dystrophy). Despite the importance of this system in human health and disease, our understanding of its molecular mechanisms is limited. We propose to combine X-ray crystallography, solution small angle X-ray scattering (SAXS) and biochemical approaches to study the human protein-DNA assemblies that are key intermediates in the lesion recognition and excision steps of MMR. We are fortunate that the human MMR pathway has now reached the maturity of analysis that obtaining the protein components and their crystals is now in hand and can be studied directly. We have already determined crystal structures of human MutS[unreadable] DNA lesion recognition complexes. In Aim 1 we propose to determine crystal structures of Exo1 and MutS[unreadable]. In Aim 2 we investigate substrate recognition, specificity and ATP-dependent conformational transitions of MMR components. In Aim 3 we study two key multi-component assemblies essential to the recognition and excision process. Together the aims will contribute to furthering our understanding of the molecular basis of MMR-associated human diseases.